Improved accuracy of DNA quantitation using a chemistry model-based method for estimating nucleic acid quantities in PCR amplified samples
Abstract number: P1826
Archer B., Dobbs M., Hazelo R., Exner M.
Objectives: Quantitative interpretation of a PCR result requires a mathematical algorithm or method to determine the amount of template in a sample. The method most commonly used depends on a specified threshold and a calibration plot of the fractional cycle at threshold crossing vs. the logarithm of the DNA amount in the sample. We have developed a new method based upon physical models in which parameters represent attributes of the PCR chemistry. This study sought to examine the potential of this method for determining concentrations of CMV, BKV, and EBV viruses in clinical specimens, and to compare the results with those obtained using threshold based methods. In addition, the ability of the chemistry method to assess the efficiency of individual PCR reactions was evaluated.
Methods: Analytical studies were performed to evaluate linearity, reproducibility, and accuracy of the chemistry based method in comparison with a threshold based method. For these studies, the 3M integrated cycler was used to run serial dilutions of viral DNA standards for each target being tested, and observed quantity values were compared to expected quantity values. In addition, suboptimal PCR reactions (containing inhibitory substances) were analyzed, and accuracy of quantitation in these specimens was monitored.
Results: The chemistry model-based method was found to be both accurate and precise over a very wide dynamic range of template concentrations, with a linear range beyond what was observed for the threshold based model. The chemistry method also was able to provide a more accurate quantification of DNA in samples for which PCR conditions were not optimal and replication efficiency was reduced.
Conclusions: By eliminating the assumptions required by threshold methods of quantitation and automatically accounting for replication efficiency in each reaction, the chemistry model-based algorithm provides more accurate quantity determinations from clinical specimens. Using this method will improve performance of diagnostic tests. The estimation of parameters that represent attributes of the chemistry and an estimate of efficiency for each reaction based on these parameters will be helpful in new test development.
|Session name:||Abstracts 20th European Congress of Clinical Microbiology and Infectious Diseases|
|Location:||Vienna, Austria, 10 - 13 April 2010|
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